Chemically-enhanced primer compositions, methods and kits

ABSTRACT

A composition is provided comprising a negatively charged group, an oligonucleotide sequence and at least none or one nuclease-resistant linkage group to form a chemically-enhanced primer. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the primer as well as a method of preparing DNA for sequencing and a method of sequencing DNA and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition under conditions in which excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims a priority benefit under 35 U.S.C. §119(e) fromU.S. Patent Application No. 61/407,899, filed Oct. 28, 2010 and PatentApplication No. 61/408,553, filed Oct. 29, 2010, which are incorporatedherein by reference.

FIELD

The present teachings pertain to chemically modified oligonucleotidesequence primer compositions and methods for sequencing DNA and fragmentanalysis. The teachings also relate to compositions for preparing,fragment analysis and sequencing of nucleic acids such as cDNA and DNA.

BACKGROUND

A standard polymerase chain reaction (PCR)/sequencing workflow generallyincludes five steps requiring reagent addition: an initial PCR step, aPCR clean-up step, a sequencing step, a sequencing cleanup step, andelectrophoresis. The PCR step involves amplification of a templatepolynucleotide using amplification primers and a thermo-stable DNApolymerase enzyme. The PCR cleanup step is commonly done by the additionof exonuclease I and alkaline phosphatase, followed by incubation, andsubsequent heat-inactivated to inactivate the enzymes. A standardPCR/sequencing workflow is illustrated in FIG. 1A.

A typical PCR reaction uses an excess of amplification primers, someprimers remain unincorporated upon completion of the PCR reaction. Thisnecessitates removal of the excess primers before proceeding to asequencing reaction, because the excess amplification primers willinterfere with the subsequent sequencing reaction. The PCR reactionfurthermore contains an excess of dNTPs that can interfere with thesubsequent sequencing reaction. The hydrolytic properties of exonucleaseI which degrades single-stranded DNA present in the PCR mixture allowsthe amplification product (amplicon) to be used more efficiently insubsequent sequencing applications. The enzyme activity of alkalinephosphatase dephosphorylates free dNTPs remaining from the PCR reaction.After an appropriate incubation period, the exonuclease I and alkalinephosphatase enzymes are heat inactivated before adding sequencingprimer, dNTPs, and dye-labeled ddNTPs; otherwise the enzymes woulddegrade these reagents and the sequencing reaction products.

Without adequate exonuclease I treatment to remove excess PCRamplification primers, aberrant sequence ladders can be generated. Anexcess of dNTPs can produce a weak sequencing signal and/or shortsequence reads. The need to obtain high quality sequence results at base1 from the sequencing primer is also often difficult. The transitionfrom amplification to efficient sequencing has made high quality 5′sequence resolution and clean-up of unincorporated dNTPs andamplification primers a priority to obtain clean sequencing results.

Resolution of nucleic acid sequence near the sequencing primer has beendifficult to obtain without sacrificing throughput residence time duringelectrophoresis with POP7™ polymer. Adjustments in the type of mobilitysystem, for example, using the POP6™ polymer matrix, adjustingdenaturing conditions and temperature can improve resolution but alwaysat the expense of increased electrophoresis time as POPE polymerrequires longer electrophoresis time. Difficulties in removal ofunincorporated reactants and long residence time when performingsize-dependent mobility separation contribute to inefficiencies innucleic acid sequencing. A need exists for improved methods for thePCR/sequencing and PCR/fragment analysis workflow and sequenceresolution following PCR amplification.

SUMMARY

According to various embodiments, the present teachings provide acomposition for a chemically-enhanced primer. According to variousembodiments, the primer can comprise a negatively charged moiety, anoligonucleotide sequence and a nuclease-resistant linkage. In variousembodiments the primer can be used in fragment analysis, sequencingnucleic acid and for improving resolution, PCR through sequencingworkflows with POP-7™ polymer on capillary electrophoresis instrumentssuch as those manufactured by Applied Biosystems (Foster City, Calif.).

According to various embodiments, the present teachings provide acomposition for sequencing nucleic acid. According to variousembodiments, the composition can comprise a composition comprising achemically-enhanced primer comprising an oligonucleotide sequence, anegatively charged moiety (NCM) and at least one nuclease-resistantlinkage. In various embodiments the composition can further comprise apolymerase, a nuclease, deoxynucleotide triphosphates (dNTPs), anddideoxynucleotide triphosphates (ddNTPs) and at least one dye-label. Invarious embodiments of the method, the composition can be added in onestep directly to a PCR reaction product, without first removing excessPCR amplification primers from the PCR reaction product.

According to various embodiments, the present teachings relate to amethod of preparing DNA for sequencing, a method of sequencing DNA, anda composition for sequencing DNA. The teachings provide a method ofPCR/sequencing (including cycle sequencing) that can be quicker andsimpler, and require fewer steps, than traditional methods. The methodsof the present teachings utilize a chemically-enhanced primer incombination with nuclease, which can reduce sequence noise and removeundesired sequence priming. The present teachings further provide acomposition for DNA sequencing that can be used with such a method.

According to various embodiments, the present teachings disclose amethod of preparing DNA for sequencing. In some embodiments, the DNApreparation method can eliminate at least one reagent addition step usedin conventional PCR/cycle sequencing, thereby reducing the number ofprocessing steps.

According to various embodiments, a method of preparing DNA forsequencing is provided that can comprise amplifying DNA under conditionsto produce amplification reaction products, the amplification reactionproducts comprise excess amplification primer, and contacting theamplification reaction products with a reaction mixture comprising anuclease and a chemically-enhanced sequencing primer, under conditionsin which the excess amplification primer is degraded by the nuclease.According to various embodiments, the chemically-enhanced primer isessentially non-degraded under such conditions. In some embodiments, theexcess amplification primer can comprise inter-nucleotide phosphodiesterbonds that are susceptible to nuclease cleavage. In some embodiments thechemically-enhanced primer can comprise at least one inter-nucleotidenuclease-resistant linkage, including but not limited to at least onephosphorothioate bond that is not susceptible to nuclease cleavage.

The present teachings further provide a method of sequencing DNA thatcan generate clean, clear and accurate sequencing data by a simplerworkflow compared to conventional methods, and that requires less time.According to various embodiments, a DNA sequencing method is providedthat can comprise adding a sequencing reaction mix directly to acompleted PCR amplification reaction, without first performing aseparate cleanup step; that is, without first removing excess PCRamplification primers by the addition of a nuclease and completing anuclease inactivation step, followed by a second addition of sequencingprimers and reagents.

According to various embodiments, a method of sequencing DNA is providedthat can comprise amplifying DNA in a first reaction mixture comprisingnuclease-sensitive amplification primers to form amplified DNA,contacting the first reaction mixture with a second reaction mixturecomprising a nuclease and a chemically-enhanced primer under conditionsin which the nuclease-sensitive amplification primers are degraded bythe nuclease, inactivating the nuclease, and causing the amplified DNAto serve as template in a sequencing reaction under conditions in whichthe chemically-enhanced primer primes the sequencing reaction.

The present teachings further provide a system for sequencing DNA thatcan comprise amplifying DNA in a first reaction mixture comprisingnuclease sensitive amplification primers to form amplified DNA,contacting said first reaction mixture of the amplifying step with asecond reaction mixture comprising a nuclease and a chemically-enhancedprimer, under conditions in which the nuclease sensitive amplificationprimers are degraded by the nuclease; inactivating the nuclease andcausing the amplified DNA to react in a sequencing reaction underconditions in which the chemically-enhanced primer primes saidsequencing reaction; and identifying a nucleotide base sequence of theamplified DNA by mobility-dependent separation of sequencing reactionproducts.

The present teachings further provide a kit. In various embodiments thekit comprises a chemically-enhanced primer comprising a negativelycharged group, an oligonucleotide sequence and a nuclease resistantmoiety. In further embodiments, the kit can have at least one of ainstructions for use, a nuclease, a sufficient quantity of enzyme forsequencing or fragment analysis, buffer to facilitate the sequencing orfragment analysis, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTPfor strand extension during sequencing or fragment analysis, ddNTPs, adye-label, loading solution for preparation of the sequenced or fragmentanalyzed material for electrophoresis, genomic DNA as a templatecontrol, a size marker to insure that materials migrate as anticipatedin the separation medium, and a protocol and manual to educate the userand limit error in use.

Various patents, patent applications, and other publications arereferred to herein, all of which are incorporated herein in theirentireties by reference. In addition, the following standard referenceworks are incorporated herein by reference: Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., edition as of October 2007;Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual,3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.In the event of a conflict between the instant specification and anydocument incorporated by reference, the specification shall control, itbeing understood that the determination of whether a conflict orinconsistency exists is within the discretion of the inventors and canbe made at any time.

Additional features and advantages of the present teachings will beevident from the description that follows, and in part will be apparentfrom the description, or can be learned by practice of the presentteachings. It is to be understood that both the foregoing summary andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the present teachingswithout limiting the present teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification exemplify embodiments disclosed and, togetherwith the description, serve to explain and illustrate principles ofdisclosed embodiments. Specifically:

FIG. 1 is a diagrammatic representation of a standard PCR/cyclesequencing workflow; with five steps in FIG. 1A and four steps in FIG.1B, the disclosed improved workflow.

FIG. 2 illustrates an exonuclease 1-resistant oligonucleotide having anuclease-resistant linkage at the terminal 3′ end, according to variousembodiments.

FIG. 3A illustrates a chemically-enhanced primer consisting of(C3)₁₀-M13*(Forward).

FIG. 3B illustrates a chemically-enhanced primer consisting of (C3)₈-M13(Forward).

FIG. 3C illustrates a chemically-enhanced primer consisting of (C3)₉-M13(Forward).

FIG. 3D illustrates a chemically-enhanced primer consisting of (C3)₅-M13(Forward).

FIG. 3E illustrates a chemically-enhanced primer consisting of(C3)₆-trebler-M13 (Forward).

FIG. 3F illustrates a chemically-enhanced primer consisting of(C3)₃-Long trebler-M13 (Forward).

FIG. 3G illustrates a chemically-enhanced primer consisting of(C₃)₈-treb-M13 (Forward).

FIG. 3H illustrates a chemically-enhanced primer consisting of(C3)₁₅-M13* (Forward), * indicates a phosphorothioate linkage.

FIG. 3I illustrates a chemically-enhanced primer consisting of(C3)₁₅-M13* (Forward), * indicates a phosphorothioate linkage.

FIG. 3J illustrates a chemically-enhanced primer consisting of(C3)₁₈-M13* (Forward), * indicates a phosphorothioate linkage

FIG. 4A-4B illustrates a chemically-enhanced primer consisting of(C3)₁₅-gene specific primer oligonucleotide sequence* (Forward) or auniversal primer oligonucleotide sequence* (Forward), respectively,indicates a phosphorothioate linkage.

FIG. 4C illustrates a chemically-enhanced primer consisting of(C3)₁₅-oligonucleotide sequence (Forward).

DETAILED DESCRIPTION

To facilitate understanding of the present teachings, the followingdefinitions are provided. It is to be understood that, in general, termsnot otherwise defined are to be given their ordinary meanings ormeanings as generally accepted in the art.

As used herein, the term “PCR/cycle sequencing” refers to a method fordetermining a nucleotide sequence of DNA by PCR amplifying the DNA,followed by sequencing reactions repeated (or cycled) several times.This cycling is similar to PCR because the sequencing reaction isallowed to proceed at 42° C.-55° C., then stopped at 95° C., and startedagain at 42° C.-55° C., and uses a thermostable DNA polymerase.

As used herein, the term “phosphorothioate linkage” refers to aninter-nucleotide linkage comprising a sulfur atom in place of anon-bridging oxygen atom within the phosphate linkages of a sugarphosphate backbone. The term phosphorothioate linkage refers to bothphosphorothioate intra-nucleotide linkages and phosphorodithioateinter-nucleotide linkages. A “phosphorothioate linkage at a terminal 3′end” refers to a phosphorothioate linkage at the 3′ terminus, that is,the last phosphate linkage of the sugar phosphate backbone at the 3′terminus. A phosphorothioate linkage at a terminal 3′ end is illustratedin FIG. 2.

As used herein, the term “phosphodiester linkage” refers to thelinkage—PO₄— which is used to link nucleotide monomers. Phosphodiesterlinkages as contemplated herein are linkages found innaturally-occurring DNA.

As used herein, the term “nuclease-resistant linkage” refers to anoligonucleotide sequence, such as a primer, that is resistant todigestion in the 3′ to 5′ direction by nuclease. Phosphorothioate andboronophosphate linkages are two examples of nuclease-resistantlinkages. The examples are not to be construed as limited to just theseexamples.

As used herein, the term “primer” refers to an oligonucleotide,typically between about 10 to 100 nucleotides in length, capable ofselectively binding to a specified target nucleic acid or “template” byhybridizing with the template. The primer can provide a point ofinitiation for template-directed synthesis of a polynucleotidecomplementary to the template, which can take place in the presence ofappropriate enzyme(s), cofactors, substrates such as nucleotides andoligonucleotides and the like.

As used herein, the term “chemically-enhanced primer” refers to a primerthat can have a negatively charged moiety at a terminal 5′ end of theprimer or within the primer. The primer can also include anuclease-resistant linkage at the last phosphate linkage of the sugarphosphate backbone at the 3′ terminus.

As used herein, the term “sequencing primer” refers to anoligonucleotide primer that is used to initiate a sequencing reactionperformed on a nucleic acid. The term “sequencing primer” refers to botha forward sequencing primer and to a reverse sequencing primer.

As used herein, the term “extension primer” refers to anoligonucleotide, capable of annealing to a nucleic acid region adjacenta target sequence, and serving as an initiation primer for elongation ofthe oligonucleotide by using the target sequence as the complementarytemplate for nucleotide extension under suitable conditions well knownin the art. Typically, a sequencing reaction employs at least oneextension primer or a pair of extension primers. The pair would includean “upstream” or “forward” primer and a “downstream” or “reverse”primer, which delimit a region of the nucleic acid target sequence to besequenced.

As used herein, the term “amplification primer” refers to anoligonucleotide, capable of annealing to an RNA or DNA region adjacent atarget sequence, and serving as an initiation primer for nucleic acidsynthesis under suitable conditions well known in the art. Typically, aPCR reaction employs a pair of amplification primers including an“upstream” or “forward” primer and a “downstream” or “reverse” primer,which delimit a region of the RNA or DNA to be amplified.

As used herein, the term “tailed primer” or “tailed amplificationprimer” refers to a primer that includes at its 3′ end a sequencecapable of annealing to an RNA or DNA region adjacent a target sequence,and serving as an initiation primer for DNA synthesis under suitableconditions well known in the art. The primer includes its 5′ end asequence capable of annealing to a sequencing primer, for example, anoligonucleotide sequence, an universal sequencing primer, agene-specific primer, primer and the like.

As used herein, the term “amplifying” refers to a process whereby aportion of a nucleic acid is replicated. Unless specifically stated,“amplifying” refers to a single replication or to an arithmetic,logarithmic, or exponential amplification.

As used herein, the term “target amplicon” refers to an amplificationproduct having the target sequence of interest and resulting form anamplification reaction, e.g., a polymerase chain reaction (PCR).

As used herein, the terms “extend”, “extension” and “extending” are usedinterchangeably and refer to a process whereby an oligonucleotide isincreased in length at the 3′ end according to a template target nucleicacid sequence. Unless specifically stated, “extend” refers to a singleexpansion or to a plurality of parallel or multiple expansions of atarget or multiple target nucleic acid target sequences.

As used herein, the term “determining a nucleotide base sequence” or theterm “determining information about a sequence” encompasses “sequencedetermination” and also encompasses other levels of information such aseliminating one or more possibilities for a sequence. It is noted thatperforming sequence determination of a polynucleotide typically yieldsequivalent information regarding the sequence of a perfectlycomplementary (100% complementary) polynucleotide and thus is equivalentto sequence determination performed directly on a perfectlycomplementary polynucleotide.

The term “nucleic acid sequence” as used herein can refer to the nucleicacid material itself and is not restricted to the sequence information(i.e. the succession of letters chosen among the five base letters A, C,G, T, or U) that biochemically characterizes a specific nucleic acid,for example, a DNA or RNA molecule. Nucleic acids shown herein arepresented in a 5′→3′ orientation unless otherwise indicated.

The term “mobility-dependent separation” as used herein can refer to theseparation of nucleic acid fragments due to the charge and sizeassociated with the fragment.

The term “fluorescent dye” as used herein refers to moieties that absorblight energy at a defined excitation wavelength and emit light energy ata different wavelength. Preferably the fluorescent dyes selected for useare spectrally resolvable. As used herein, “spectrally resolvable” meansthat the dyes can be distinguished on the basis of their spectralcharacteristics, particularly fluorescence emission wavelength, underconditions of operation. For example, the identity of the one or moreterminal nucleotides can be correlated to a distinct wavelength ofmaximum light emission intensity, or perhaps a ratio of intensities atdifferent wavelengths.

As used herein, the terms “polynucleotide”, “nucleic acid”, or“oligonucleotide” refers to a linear polymer of nucleosides (includingdeoxyribonucleosides, ribonucleosides, or analogs thereof) joined byinter-nucleosidic linkages. Whenever a polynucleotide such as anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotesdeoxythymidine, unless otherwise noted. The letters A, C, G, and T canbe used to refer to the bases themselves, to nucleosides, or tonucleotides comprising the bases, as is standard in the art. Innaturally occurring polynucleotides, the inter-nucleoside linkage istypically a phosphodiester bond, and the subunits are referred to as“nucleotides.” Oligonucleotide primers comprising other inter-nucleosidelinkages, such as phosphorothioate linkages, are used in certainembodiments of the teachings. It will be appreciated that one or more ofthe subunits that make up such an oligonucleotide primer with anon-phosphodiester linkage can not comprise a phosphate group. Suchanalogs of nucleotides are considered to fall within the scope of theterm “nucleotide” as used herein, and nucleic acids comprising one ormore inter-nucleoside linkages that are not phosphodiester linkages arestill referred to as “polynucleotides”, “oligonucleotides”, etc.

As used herein “sequence determination”, “determining a nucleotide basesequence”, “sequencing”, and like terms includes determination ofpartial as well as full sequence information. That is, the term includessequence comparisons, fingerprinting, and like levels of informationabout a target polynucleotide, as well as the express identification andordering of each nucleoside of the target polynucleotide within a regionof interest. In certain embodiments, “sequence determination” comprisesidentifying a single nucleotide, while in other embodiments more thanone nucleotide is identified. Identification of nucleosides,nucleotides, and/or bases are considered equivalent herein. It is notedthat performing sequence determination on a polynucleotide typicallyyields equivalent information regarding the sequence of a perfectlycomplementary polynucleotide and thus is equivalent to sequencedetermination performed directly on a perfectly complementarypolynucleotide.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, primerset(s), etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits can include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to a delivery system comprising two or more separate containersthat each contain a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Indeed,any delivery system comprising two or more separate containers that eachcontains a subportion of the total kit components are included in theterm “fragmented kit.” In contrast, a “combined kit” refers to adelivery system containing all of the components of a reaction assay ina single container (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

As will be appreciated by one of ordinary skill in the art, referencesto templates, oligonucleotides, primers, etc., generally meanpopulations or pools of nucleic acid molecules that are substantiallyidentical within a relevant region rather than single molecules. Forexample, a “template” generally means a plurality of substantiallyidentical template molecules; a “primer” generally means a plurality ofsubstantially identical primer molecules, and the like.

Cycle sequencing involves adding to a target nucleic acid or anamplification product thereof, sequencing primer, deoxynucleotidetriphosphates (dNTPs), dye-labeled chain terminating nucleotides (e.g.,dideoxynucleotide triphosphates (ddNTPs-dyes)), and DNA polymerase,followed by thermal cycle sequencing. Standard cycle sequencingprocedures are well established. Cycle sequencing procedures aredescribed in more detail, for example, in U.S. Pat. No. 5,741,676, andU.S. Pat. No. 5,756,285, each herein incorporated by reference in itsentirety. In certain embodiments, “cycle sequencing” comprises dNTPS, asequencing primer (labeled or not), ddNTPs (labeled or not) and DNApolymerase as known to one of skill in the art. It is noted that alabeled sequencing primer can provide fragment analysis informationand/or determination of the sequence of a target nucleic acid oramplification product thereof.

According to various embodiments of the present teachings, provided is achemically-enhanced primer comprising an oligonucleotide sequence, anegatively charged moiety (NCM) and at least one nuclease-resistantlinkage. In some embodiments the at least one nuclease-resistant linkageincludes but is not limited to at least one phosphorothioate linkage(PS) or at least one boronophosphate linkage. In other embodiments thenuclease-resistant linkage is not present in the chemically-enhancedprimer. The primer can be used to prime a target nucleic acid in asequencing reaction, herein referred to as a chemically-enhancedsequencing primer or for fragment analysis, herein referred to as achemically-enhanced extension primer. The oligonucleotide sequence canbe a universal primer or a gene specific nucleotide sequence. Examplesof universal primers include but are not limited to M13 (P/N 402071 and402072, Applied Biosystems), US1 (UNISEQ, PLoS Medicine 3(10)e431(2006)), T7 (P/N 402126, but without dye, Applied Biosystems), SP6 (P/N402128, but without dye, Applied Biosystems), and T3 (P/N 402127, butwithout dye, Applied Biosystems). See the ABI PRIMS® 377 DNA Sequence96-Lane Upgrade User's Manual for primer sequences. The oligonucleotidesequence can also contain a dye-label such as a fluorescent label. Invarious embodiments of the present teachings the NCM can be located atthe terminal 5′ end of the oligonucleotide sequence or within theoligonucleotide sequence. Examples of NCM include but are not limited tophosphoramidite, a (C)n spacer wherein n can be from 1-9 (available fromGlen Research), the amino acids aspartic acid and glutamic acid as wellas nucleotides and nucleotide analogs (dATP, dCTP, dGTP and dTTP). TheNCM can contain only one negatively charged monomer or a plurality ofnegatively charged moieties, for example at least five, ten, 12, 15, 18,20, 24 or more repeat units of the spacer, for example, (Cn)_(x), wherex is any integer between 1 and at least 11, at least 12, at least 15, atleast 18, at least 20, at least 24 or more Cn spacers where “n” is 3 or6, e.g., C3 spacers, C6 spacers or a combination of C3 and C6 spacers ina linear arrangement or a branched arrangement. The C3 and C6 spacersindividually or in combination can also form a branched NCM by forming adoubler or a trebler such as, for example, (C3)₃-treb-M13 or[(C3)₂-treb]-treb-M13, where the NCM is represented by (C3)₃-treb or[(C3)₂-treb]-treb and M13 represents the oligonucleotide sequence, aswould be known to one of skill in the art. The NCM can also contain adye-label such as a fluorescent label. In various embodiments at leastnone, at least one, at least two or more phosphorothioate linkages canbe at a terminal 3′ end of the oligonucleotide sequence. The presence ofat least one nuclease-resistant linkage provides resistance to digestionby 3′-5′ nucleases such as Exonuclease I (P/N MO293S New EnglandBiolabs, Ipswich, Mass.), Exo III (P/N MO206S, New England Biolabs,Ipswich, Mass.), Pfu (Promega, P/N M7741, Madison, Wis.), and DNA pol I(P/N M0209S, New England Biolabs, Ipswich, Mass.). The resistance of thechemically-enhanced primer to nuclease digestion offers the advantage ofeliminating a PCR clean-up step in the PCR to sequencing protocol.Removal of the extra non-nuclease resistant amplification primers leftover from the PCR step can be accomplished in the sequencing reactionmixture. A brief exposure of the PCR amplification reaction to thenuclease within the sequencing reaction mixture degrades thenon-nuclease resistant amplification primers followed by an inactivationof the nuclease. The chemically-enhanced primer remains available forthe sequencing reaction while the non-nuclease resistant amplificationprimers and the nuclease have been removed and inactivated,respectively.

Using the chemically-enhanced sequencing primer, it has been found thathigh quality and highly accurate nucleic acid sequence results can beobtained in about 50% less time overall for a PCR to sequencing resultsworkflow using POP7™ polymer on any of the 3130, 3730 or 3500 capillaryelectrophoresis platforms (Applied Biosystems, Foster City, Calif.) andwith results comparable to the slower POP6™ polymer. Specifically,improvement of 5′ sequence resolution to base 1 from thechemically-enhanced sequencing primer. Prior to the present teachings astandard sequencing primer using the BigDye® Terminator Kit v3.1(Applied Biosystems) with POP-7™ polymer did not provided interpretabledata the first 20-30 bases after the sequencing primer while using thePOP-6™ polymer and BigDye® Terminator Kit v1.1 was able to obtainreadable sequence by capillary electrophoresis within five bases of thesequencing primer, yet the throughput time with POP-6 polymer is slower(data not shown). The current teachings provide a NCM sequencing primerthat provided high quality bases right after the sequencing primer withPOP-7™ polymer by capillary electrophoresis and with resolutiontypically equivalent or superior in quality to BigDye® Terminator v1.1with POP-6™ polymer (see FIG. 3 of U.S. Ser. No. 61/407,899, filed Oct.28, 2011 and U.S. Ser. No. 61/408,553, filed Oct. 29, 2011). Thechemically-enhanced sequencing primer is able to run with POP-7 polymerhaving an electrophoretic run time as short as 65 minutes to generate700 high quality bases starting from the first base using a 3500 GeneticAnalyzer (Applied Biosystems). In contrast, it took 135 minutes withPOP-6 polymer to produce only 600 high quality bases. The primer of thepresent teachings in conjunction with the POP-7 polymer provided a 52%throughput increase compared to the electrophoresis time with the POP-6polymer. The throughput was increased as well as reducing hands-on-timeby eliminating a separate PCR clean-up step prior to initiation of thesequencing reaction. (FIG. 1A and FIG. 1B).

As shown in FIGS. 13-16 (presented in U.S. Ser. No. 61/407,899, filedOct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct. 29, 2011), thechemically-enhanced sequencing primer and workflow was used toinvestigate polymorphisms in Human Leukocyte Antigens (HLA). The HLAsare used for tissue and organ typing as well as tissue and organcross-matching for transplantation matching and evaluation of rejection.The SeCore® HLA-DRB1 (Invitrogen, Carlsbad, Calif.) primer set and groupspecific sequencing primers (GSSP) were used on 34 DNA samples.Sequencing reactions were performed with traditional sequencing primersand with the chemically-enhanced sequencing primers. The sequencingreaction products were electrophoresed on an Applied Biosystems 3500xl™Genetic Analyzer using POP7 polymer (Applied Biosystems). Comparison of5′ resolution and basecalling accuracy and quality was made between thetwo primers. On average the traditional sequencing primers with POP7polymer yielded high quality readable bases by 25 bases after thesequencing primer while the chemically-enhanced sequencing primersyielded high quality bases by base 5 and by base 1 in many cases andalso resulted in increased basecalling accuracy and a 40% decrease inoverall workflow time. Table 1 provides examples of the improvedsequencing quality obtained with the chemically-enhanced primers.

TABLE 1 # of edits # of edits Sample ID allele 1 DNA allele 2 DNABigDye ® Direct Secore 1 D062 DRB1*030101 DRB1*0404 0 0 2 D075DRB1*030101 DRB1*100101 0 0 3 D111 DRB1*010101 DRB1*080101 0 0 4 D115DRB1*030101 DRB1*1503 2 0 5 D125 DRB1*010101 DRB1*070101 0 0 6 D140DRB1*070101 DRB1*1311 0 0 7 D165 DRB1*110101 DRB1*1504 0 2 8 D205DRB1*010201 DRB1*1202 0 0 9 D218 DRB1*1001 DRB1*1320 1 0 1 D099DQB1*030101 DQB1*050101 4 8 2 D108 DQB1*050101 DQB1*0202 0 0 3 D113DQB1*0201 DQB1*050101 2 1 4 D116 DQB1*050301 DQB1*060101 0 0 5 D130DQB1*030302 DQB1*0502 2 2 6 D130 DQB1*030302 DQB1*0502 0 0 7 D135DQB1*0202 DQB1*030101 0 0 8 D150 DQB1*050201 DQB1*030201 1 0 9 D154DQB1*040102 DQB1*060101 0 1 10 D161 DQB1*0201 DQB1*0302 0 0 11 D168DQB1*030101 DQB1*050101 2 3 12 D181 DQB1*0301 DQB1*0501 2 3 13 F2150DQB1*0401/02 DQB1*060101.. 0 0 14 F2160 DQB1*0401/02 DQB1*060101-17 0 115 F2297 DQB1*020101-04... DQB1*060101 0 2 16 U415 DQB1*0402 DQB1*0601 00 17 U415 DQB1*0402 DQB1*0601 1 1 1 D049 DPB1*010101 DPB1*040101 0 0 2D105 DPB1*110101 DPB1*1701 0 0 3 D149 DPB1*1001 DPB1*200101 0 0 4 D157DPB1*010101 DPB1*040101 0 0 5 D161 DPB1*040101 DPB1*0601 0 0 6 D164DPB1*020102 DPB1*0601 0 0 7 D219 DPB1*040101 DPB1*0501 0 0 8 U514DPB1*0201 DPB1*0401 0 0 total edits 17 24

The results in Table 1 illustrate the relative basecalling accuracybetween the SeCore® HLA Sequence and the chemically-enhanced sequencingprimer when sequenced in both directions of exon 2 from HLA-DRB1, DQB1and DPB1 in 34 different samples. uTYPE® HLA Sequencing Software(Invitrogen) aligned the forward and reverse traces to a referencesequence and a HLA library. Basecalling accuracy was assessed by howmany base positions required manual edits to resolve discrepanciesbetween the forward, reverse and reference sequence. Thechemically-enhanced sequencing primer equaled or slightly outperformedthe existing method by requiring fewer manual edits (17) and provided asimplified workflow and faster electrophoresis time vs. 24 edits withthe traditional primers, method and workflow. The chemically-enhancedsequencing primer also improved 5′ mobility seen as 5′ C/A and A/G inDPB1, resolved shoulder problems in HLA-DQB1, and reduced C nucleotidecompression routinely observed in HLA-DRB-1 sequences to substantiallyimprove primary mixed basecalling (data not shown). Thechemically-enhanced sequencing primer and improved workflow improvedpolymorphism detection and more efficient use of allele specificsequencing primers for heterozygous ambiguity resolution resulting insignificant time savings for obtaining data that was superior in qualityto existing methods.

According to various embodiments of the present teachings, a compositionfor sequencing nucleic acid can comprise a polymerase, a nuclease, achemically-enhanced sequencing primer, dNTPs, and a chain terminator(e.g., ddNTPs). In some embodiments, the polymerase can comprise Taqpolymerase, for example AmpliTaq Gold polymerase. In some embodiments,the nuclease can comprise exonuclease I. In some embodiments, thechemically-enhanced sequencing primer can comprise at least onephosphorothioate linkage. In other embodiments, the chemically-enhancedsequencing primer can comprise a terminal 3′ end phosphorothioatelinkage. In some embodiments, the ddNTPs can comprise ddNTPs-dyes, forexample fluorescent dye-labeled ddNTPs. In some embodiments thechemically-enhanced sequencing primer can comprise a dye, for example afluorescent dye-labeled oligonucleotide and/or at least onefluorescently dye-labeled NCM moiety within the NCM compound.

According to various embodiments, the composition for sequencing nucleicacid can comprise a polymerase, for example a DNA polymerase, in anamount of from about 0.01 Unit to about 20 Units, for example, fromabout 0.1 Unit to about 1.0 Unit, or about 0.8 Unit. The composition cancomprise polymerase in an amount within a range having an upper limit offrom about 10 Units to about 20 Units and a lower limit of from about0.01 Unit to about 0.05 Unit. According to various embodiments, thecomposition can comprise a nuclease, for example exonuclease I, in anamount of from about 1 Unit to about 40 Units, for example, from about 2Units to about 15 Units, or about 10 Units. The composition can comprisenuclease in an amount within a range having an upper limit of from about10 Units to about 40 Units, and a lower limit of from about 1 Unit toabout 2 Units.

According to various embodiments, the composition for sequencing nucleicacid can comprise a chemically-enhanced sequencing primer, in an amountof from about 0.1 μM to about 20 μM, for example about 1.0 μM. Thecomposition can comprise a chemically-enhanced sequencing primer in anamount within a range having an upper limit of from about 10 μM to about20 μM and a lower limit of from about 0.05 μM to about 0.1 μM. Accordingto various embodiments, the composition can comprise dNTPs in an amountof from about 20 μM to about 5000 μM, for example, about 500 μM. Thecomposition can comprise dNTPs in an amount within a range having anupper limit of from about 2000 μM to about 5000 μM and a lower limit offrom about 20 μM to about 50 μM. According to various embodiments, thecomposition can comprise ddNTPs in an amount of from about 0.03 μM toabout 10 μM, for example about 3 μM. The composition can comprise ddNTPsin an amount within a range having an upper limit of from about 5 μM toabout 10 μM and a lower limit of from about 0.01 μM to about 0.05 μM.All molar amounts are based on final concentrations of the final volume.

According to various embodiments, the composition can comprise anon-nuclease-resistant amplification primer in an amount of from about0.1 μM to about 20 μM, for example about 1.0 μM. The composition cancomprise a non-nuclease-resistant amplification primer in an amountwithin a range having an upper limit of from about 10 μM to about 20 μMand a lower limit of from about 0.05 μM to about 0.1 μM. All molaramounts are based on final concentrations of the final volume.

According to various embodiments, the composition for sequencing nucleicacid can further comprise a PCR amplification product. In someembodiments, the PCR amplification product can comprise an amplified DNAtarget sequence. In some embodiments, the PCR amplification product cancomprise non-nuclease-resistant amplification primer(s). Thenon-nuclease-resistant amplification primer can comprise, for example,phosphodiester linkages that are sensitive to degradation byexonuclease. In some embodiments, the PCR amplification product cancomprise a target specific amplicon that incorporates nucleic acidsequence capable of annealing to a universal primer.

According to various embodiments of the present teachings, a method ofpreparing a nucleic acid for sequencing can comprise a step ofamplifying the nucleic acid under conditions to produce amplificationreaction products. The nucleic acid can be amplified using, for example,polymerase chain reaction (PCR). The nucleic acid can also be amplifiedusing other methods such as, for example, multiple strand displacementamplification, helicase displacement amplification, nick translation, Qbeta replicase amplification, rolling circle amplification, and otherisothermal amplification methods.

According to various embodiments, the nucleic acid to be amplified cancomprise, for example, RNA, DNA, cDNA, genomic DNA, viral DNA, plasmidDNA, recombinant DNA, amplicon DNA, synthetic DNA or the like. Templatemolecules can be obtained from any of a variety of sources. For example,DNA as a template molecule can be isolated from a sample, which can beobtained or derived from a subject. The word “sample” is used in a broadsense to denote any source of a template on which sequence determinationis to be performed. The phrase “derived from” is used to indicate that asample and/or nucleic acids in a sample obtained directly from a subjectcomprising nucleic acid can be further processed to obtain templatemolecules.

The source of a sample can be of any viral, prokaryotic,archaebacterial, or eukaryotic species or a synthetic species. Incertain embodiments the source can be a human. The sample can comprise,for example, embryonic, cultured cells, tissues or organs, bone, tooth,organ, tissue, preserved, e.g., formalin-fixed paraffin embed (PFPE)organ or tissue, degraded, mummified, or tissue including blood oranother body fluid containing cells, such as sperm, a biopsy sample, orthe like. Mixtures of nucleic acids from different samples and/orsubjects can be combined. Samples can be processed in any of a varietyof ways. Nucleic acids can be isolated, purified, and/or amplified froma sample using known methods.

Amplifying nucleic acid can typically result in a reaction product thatcomprises excess amplification primer and an amplicon (also referred toas an amplification product) that comprises a target nucleic acid.According to various embodiments, a method of preparing nucleic acid forsequencing can comprise removing excess amplification primer from thereaction product. In some embodiments, the amplification primer can beremoved, for example, by adding a nuclease enzyme and providingappropriate conditions for the nuclease to degrade the amplificationprimer. In some embodiments, the amplification primer can be removed bycontacting the amplification reaction product with a reaction mixturecomprising a nuclease enzyme. Nucleases suitable for use in the subjectmethods preferentially degrade single-stranded polynucleotides overdouble-stranded polynucleotides, thus destroying excess primers whileleaving intact double-stranded amplicons available for sequencing insubsequent steps. In various embodiments, the nuclease enzyme cancomprise, for example, exonuclease I. Exonuclease I can be obtained fromvarious commercial suppliers, for example from USB Corp., Cleveland,Ohio. Appropriate reaction conditions can include, for example, optimaltime, temperature, and buffer parameters to provide for nuclease enzymeactivity. In some embodiments, for example, excess amplification primercan be degraded by adding exonuclease I to the amplification reactionproduct and incubating at about 37° C. for about 10 to about 30 min.Exonuclease I can hydrolyze single-stranded DNA in a 3′→5′ direction.

According to various embodiments of a method for preparing a nucleicacid, a reaction mixture can further comprise a chemically-enhancedsequencing primer. The chemically-enhanced sequencing primer can beessentially non-degraded by a reaction mixture comprising a nuclease,for example, exonuclease I, under reaction conditions at which excessamplification primer can be degraded by the nuclease. By “essentiallynon-degraded” it is intended that any degradation that takes place ofthe chemically-enhanced sequencing primer is not of a level thatsignificantly interferes with the process employed to generatesequencing and/or fragment analysis data in the subsequent sequencingreactions or fragment analysis reactions.

According to various embodiments, the chemically-enhanced sequencingprimer can comprise an oligonucleotide sequence. In some embodiments,the chemically-enhanced sequencing primer can comprise one of morenuclease-resistant internucleotide linkage(s). For example, theinternucleotide linkage may be a phosphorothioate linkage. In someembodiments, the chemically-enhanced sequencing primer can comprise anuclease-resistant internucleotide linkage at a terminal 3′ end, at aterminal 5′ end, and/or at one or more internal linkage sites. In someembodiments, the nuclease resistant internucleotide linkage is at leastone phosphorothioate linkage. Chemically-enhanced sequencing primerswere synthesized having one or two phosphorothioate linkages on theterminal 3′ end to protect the chemically-enhanced sequencing primersfrom exonuclease I digestion. The Sp stereoisomer can protect the primerfrom exonuclease I digestion but the Rp stereoisomer was found toprovide no protection from exonuclease I digestion (data not shown).

The use of a chemically-enhanced sequencing primer having theconfiguration (C3)₈-trebler-M13Rev with either one or twophosphorothioate linkages was evaluated in the sequencing of twotemplates, #30 and #122. It was found that in both templates the firstbase (A) is split into two peaks with the primer having onephosphorothioate linkage, and the first two bases (A and T) were splitwith the primer having two phosphorothioate linkages because thestereoisomers have slightly different mobilities. For the primer withone phosphorothioate linkage, there is either oligo-Sp or oligo-Rp. Inthe case of two phosphorothioate linkages on the terminal 3′ end of theprimer, there were four possible isomers (oligo-Sp-Sp, oligo-Rp-Sp,oligo-Rp-Rp and oligo-Sp-Rp) on the primer with two phosphorothioatelinkages. It was found that as with plasmid sequencing, mobilitydifferences were caused by the Sp and Rp stereoisomers resulted in thesplitting of the first two peaks.

In some embodiments, the chemically-enhanced primer can comprise anegatively charged group/compound/molecule (NCM). For example, the NCMcan be located at the terminal 5′ end of the oligonucleotide sequence orwithin the oligonucleotide sequence. Examples of NCM are disclosed above(FIGS. 3A-3J, 4A-4C). The NCM can be attached to both a non-dye-labeledoligonucleotide sequence which functions as the primer sequence as wellas to a dye-labeled oligonucleotide sequence which function as theprimer sequence. In some embodiments, the dye can be attached to anucleotide or nucleotide analog of the oligonucleotide sequence or tothe NCM as would be known to one of skill in the art.

The movement of DNA fragments under an electric field depends on thecharge to mass ratio. The DNA fragment can move differently by adding aNCM. Numerous NCM configurations and compositions attached to sequencingprimers were evaluated for their ability to change the amplifiedsequencing fragments mobility under an electric field. Figures FIGS.3A-3J, 4A-4C illustrate exemplary M13 oligonucleotide sequence primersand gene-specific primers with various NCMs. The chromatogramsillustrating the results of sequencing reactions with the exemplaryNCM+oligonucleotide sequence primer structures with a variety oftemplates can be found in FIGS. 3-12 and 16 of U.S. Ser. No. 61/407,899,filed Oct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct. 29, 2011).

In various embodiments, “analogs” in reference to nucleosides/tidesand/or polynucleotides comprise synthetic analogs having modifiednucleobase portions, modified pentose portions and/or modified phosphateportions, and in the case of polynucleotides, modified internucleotidelinkages, as described generally elsewhere (e.g., Scheit, NucleotideAnalogs (John Wiley, New York, (1980); Englisch, Angew. Chem. Int. ed.Engl. 30:613-29 (1991); Agrawal, Protocols for Polynucleotides andAnalogs, Humana Press (1994)). Generally, modified phosphate portionscomprise analogs of phosphate wherein the phosphorous atom is in the +5oxidation state and one or more of the oxygen atoms is replaced with anon-oxygen moiety e.g., sulfur. Exemplary phosphate analogs include butare not limited to phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, boronophosphates, includingassociated counterions, e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions arepresent. Exemplary modified nucleobase portions include but are notlimited to 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne,isocytosine, isoguanine, 2-thiopyrimidine, and other like analogs.Particularly preferred nucleobase analogs are iso-C and iso-G nucleobaseanalogs available from Sulfonics, Inc., Alachua, Fla. (e.g., Benner, etal., U.S. Pat. No. 5,432,272). Exemplary modified pentose portionsinclude but are not limited to 2′- or 3′-modifications where the 2′- or3′-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy,allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino oralkylamino, fluoro, chloro, bromo and the like. Modified internucleotidelinkages include phosphate analogs, analogs having achiral and unchargedintersubunit linkages (e.g., Sterchak, E. P., et al., Organic Chem,52:4202 (1987)), and uncharged morpholino-based polymers having achiralintersubunit linkages (e.g., U.S. Pat. No. 5,034,506). A particularlypreferred class of polynucleotide analogs where a conventional sugar andinternucleotide linkage has been replaced with a 2-aminoethylglycineamide backbone polymer is peptide nucleic acid (PNA) (e.g., Nielsen etal., Science, 254:1497-1500 (1991); Egholm et al., J. Am. Chem. Soc.,114: 1895-1897 (1992)).

According to various embodiments, the chemically-enhanced primer cancomprise a universal primer, selected from US1, M13, T7, SP6, T3, orother sequencing primers as would be known to one of skill in the art.For example, an M13 universal forward primer, an M13 universal reverseprimer, or the like. In other embodiments, the chemically-enhancedprimer can comprise an oligonucleotide sequence or a specific genespecific oligonucleotide sequence, to the target nucleic acid sequencefor which the nucleic acid sequence was to be determined. Theoligonucleotide primer sequence can be the identical primer sequence asthe sequence of the amplification primer used to generate the PCRamplification product having the amplified nucleic acid target sequenceand/or DNA target sequence.

While embodiments of a method for preparing nucleic acid for sequencingcan comprise using a phosphorothioated sequencing primer, and theteachings disclosed herein exemplify using a sequence primer having aNCM at or near the terminal 5′ end and a phosphorothioated terminal 3′end of an oligonucleotide sequence, other types of chemically-enhancedprimers can be utilized within the scope of the present teachings. Forexample, a nuclease resistant sequencing primer can comprise an alkylphosphonate monomer, RO—P(═O)(—Me)(—OR), such as dA-Me-phosphonamidite,and/or a triester monomer, RO—P(═O)(—OR′)(—OR), such asdA-Me-phosphoramidite (available from Glen Research, Sterling, Va.),and/or a locked nucleic acid monomer (available from Exiqon, Woburn,Mass.), and/or a boranophosphate monomer, RO—P(—BH₃)(═O) (—OR), asdescribed by Shaw, Barbara Ramsey, et al., in “Synthesis ofBoron-Containing ADP and GDP Analogues: Nucleoside5′-(P-Boranodisphosphates)”, Perspectives in Nucleoside and Nucleic AcidChemistry, pg. 125-130, (2000), or the like.

According to various embodiments, the amplification reaction productscan comprise a target amplicon. In some embodiments, the target ampliconcan comprise a result of PCR amplification from amplification primers.In some embodiments, the target amplicon can comprise double strandedDNA. In some embodiments, the target amplicon can comprise singlestranded DNA.

According to various embodiments, the amplification primers can comprisetailed primers. The tailed primers can be used, for example, to generatea target specific amplicon that incorporates nucleic acid sequencecapable of annealing to a universal primer or a gene specific primer.

According to various embodiments, a method for preparing nucleic acidfor sequencing can comprise inactivating a nuclease after excess primeris degraded by the nuclease. In some embodiments, the nuclease can beinactivated by heating. For example, the nuclease can beheat-inactivated by heating to a temperature of from about 80° C. toabout 90° C. for about 15 minutes. In various embodiments, theinactivation of the nuclease can occur within the vesicle and in thesame reaction step as the sequencing reaction as shown in FIG. 1B in theCycle Seq. (cycle sequencing) step.

According to various embodiments of the present teachings, templates tobe sequenced can be synthesized by PCR in individual aqueouscompartments (also called “reactors”) of an emulsion. In someembodiments, the compartments can each contain a particulate supportsuch as a bead having a suitable first amplification primer attachedthereto, a first copy of the template, a second amplification primer,and components needed for a PCR reaction (for example nucleotides,polymerase, cofactors, and the like). Methods for preparing emulsionsare described, for example, in U.S. Pat. No. 6,489,103 B1, U.S. Pat. No.5,830,663, and in U.S. Patent Application Publication No. US2004/0253731. Methods for performing PCR within individual compartmentsof an emulsion to produce clonal populations of templates attached tomicroparticles are described, for example, in Dressman, D., et al, Proc.Natl. Acad. Sci., 100(15):8817-8822, 2003, and in PCT publicationWO2005010145. All of the patents, applications, publications, andarticles described herein are incorporated in their entireties byreference.

According to various embodiments, a method for sequencing nucleic acidcan comprise amplifying nucleic acid in a first reaction mixturecomprising nuclease sensitive amplification primers to form amplifiednucleic acid, contacting the first reaction mixture with a secondreaction mixture comprising a nuclease and a chemically-enhanced primer,under conditions in which the nuclease sensitive amplification primersare degraded by the nuclease, and inactivating the nuclease and causingthe amplified nucleic acid to react in a sequencing reaction underconditions in which the chemically-enhanced primer primes the sequencingreaction. According to various embodiments, results can be obtainedbased on the sequencing reaction and a nucleotide base sequence of theamplified nucleic acid can be determined based on the results. Accordingto various embodiments, the nucleotide base sequence can be determinedby a mobility-dependent separation of the sequencing reaction products.According to various embodiments, the amplifying can be by polymerasechain reaction amplification.

According to various embodiments, the second reaction mixture canfurther comprise dNTPs, ddNTPs, a dye-label, and a thermo-stable DNApolymerase. In some embodiments, each of the ddNTPs can be labeled witha different fluorescent dye (ddNTP-dye). For example, the ddNTPs cancomprise BigDye ddNTPs, available from Applied Biosystems, Foster City,Calif. In some embodiments, the chemically-enhanced primer can belabeled with a fluorescent dye. The label can be attached to theoligonucleotide sequence and/or the NCM region of thechemically-enhanced primer. The thermo-stable DNA polymerase cancomprise, for example, a DNA polymerase known to one of skill in theart. In some embodiments, the sequencing reaction can comprise a thermalcycle sequencing reaction.

A variety of nucleic acid polymerases may be used in the methodsdescribed herein. For example, the nucleic acid polymerizing enzyme canbe a thermostable polymerase or a thermally degradable polymerase.Suitable thermostable polymerases include, but are not limited to,polymerases isolated from Thermus aquaticus, Thermus thermophilus,Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, andThermotoga maritima. Suitable thermodegradable polymerases include, butare not limited to, E. coli DNA polymerase I, the Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNApolymerase, and others. Examples of other polymerizing enzymes that canbe used in the methods described herein include T7, T3, SP6 RNApolymerases and AMV, M-MLV and HIV reverse transcriptases.

Non-limiting examples of commercially available polymerases that can beused in the methods described herein include, but are not limited to,TaqFS®, AmpliTaq® CS (Applied Biosystems), AmpliTaq FS (AppliedBiosystems), AmpliTaq Gold® (Applied Biosystems), Kentaq1 (AB Peptide,St. Louis, Mo.), Taquenase (ScienTech Corp., St. Louis, Mo.),ThermoSequenase (Amersham), Bst polymerase, Vent_(R)(exo⁻) DNApolymerase, Reader™Taq DNA polymerase, VENT™ DNA polymerase (New EnglandBiolabs), DEEPVENT™ DNA polymerase (New England Biolabs), PFUTurbo™ DNApolymerase (Stratagene), Tth DNA polymerase, KlenTaq-1 polymerase,SEQUENASE™ 1.0 DNA polymerase (Amersham Biosciences), and SEQUENASE 2.0DNA polymerase (United States Biochemicals).

According to various embodiments of a method for sequencing nucleicacid, the nuclease can comprise exonuclease I. The exonuclease I can besensitive to heat inactivation and can be essentially 100 percentdeactivated by heating, for example, heating at about 80° C. for about15 minutes. Other heat inactivated nucleases may be used in the subjectmethods and compositions including but not limited to Exo III, Pfu orDNA pol I.

According to various embodiments, the chemically-enhanced primercomprises at least one phosphorothioate linkage. In some embodiments,the chemically-enhanced primer comprises at least one terminal 3′ endphosphorothioate linkage. In some embodiments, as described above, thechemically-enhanced primer comprises a NCM and an oligonucleotidesequence 5′ of the phosphorothioate linkage.

The sequencing reaction products can be analyzed on a sieving ornon-sieving medium. In some embodiments of these teachings, for example,the PCR products can be analyzed by electrophoresis; e.g., capillaryelectrophoresis, as described in H. Wenz et al. (1998), GENOME RES.8:69-80 (see also E. Buel et al. (1998), J. FORENSIC SCI. 43:(1), pp.164-170)), or slab gel electrophoresis, as described in M. Christensenet al. (1999), SCAND. J. CLIN. LAB. INVEST. 59(3): 167-177, ordenaturing polyacrylamide gel electrophoresis (see, e.g., J. Sambrook etal. (1989), in MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.13.45-13.57). The separation of DNA fragments in electrophoresis isbased primarily on differential fragment size. Sequencing reactionproducts can also be analyzed by chromatography; e.g., by size exclusionchromatography (SEC). Likewise, fragment analysis can be carried in asimilar manner as would be known to the skilled artisan.

According to various embodiments a system for sequencing DNA cancomprise amplifying DNA in a first reaction mixture comprising nucleasesensitive amplification primers to form amplified DNA; contacting saidfirst reaction mixture of the amplifying step with a second reactionmixture comprising a nuclease and a chemically-enhanced primer, underconditions in which the nuclease sensitive amplification primers aredegraded by the nuclease; inactivating the nuclease and causing theamplified DNA to react in a sequencing reaction under conditions inwhich the chemically-enhanced primer primes said sequencing reaction;and identifying a nucleotide base sequence of the amplified DNA bymobility-dependent separation of sequencing reaction products. Invarious embodiments, the system for sequencing DNA, themobility-dependent separation is selected from separation by charge andseparation by size, wherein the separation by size plus charge isselected from gel electrophoresis and capillary electrophoresis andseparation by size is by a liquid gradient, and a denaturing gradientmedium.

The present teachings are also directed to kits that utilize thechemically-enhanced primer composition and methods described above. Insome embodiments, a basic kit can comprise a container having one ormore chemically-enhanced primers. A kit can also optionally compriseinstructions for use. A kit can also comprise other optional kitcomponents, such as, for example, one or more of a nuclease, asufficient quantity of enzyme for sequencing or fragment analysis,buffer to facilitate the sequencing reaction or fragment analysisreaction, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTP forstrand extension during sequencing reaction or fragment analysisreaction, ddNTPs, a dye-label, loading solution for preparation of thesequenced or fragment analyzed material for electrophoresis, genomic DNAas a template control, a size marker to insure that materials migrate asanticipated in the separation medium, and a protocol and manual toeducate the user and limit error in use. The amounts of the variousreagents in the kits also can be varied depending upon a number offactors, such as the optimum sensitivity of the process. It is withinthe scope of these teachings to provide test kits for use in manualapplications or test kits for use with automated detectors or analyzers.

Examples of the compositions and methods of the present teachings areshown below. These examples are not limiting of the present teachings,and those of ordinary skill in the art will recognize that thecomponents used in the reactions may be readily substituted withequivalent reagents known in the art.

The following Examples illustrate the ability of the chemically-enhanceprimer to provide higher resolution of a sequencing reaction and in lesstime with POP7 polymer and variants thereof, the stability of thechemically-enhance primer to exonuclease I, the incorporation of thechemically-enhance primer as a substrate for DNA polymerase, thecompatibility of exonuclease I with the sequencing reagents, and thesusceptibility of non-phosphorothioate primer to exonuclease Idigestion. The Examples further illustrate the use of tailedamplification primers along with universal phosphorothioate primers forsequencing.

Example 1

C6 spacer+Olio seq. synthesis, no phosphorothioate group: An 18 baseoligonucleotide labeled with one or more C6 spacers at the 5′ positionwas made on an ABI model 394 DNA synthesizer using standardphosphoramidite chemistry. The C6 spacer phosphoramidite was obtainedfrom Chem Genes Corp. (P/N CLP-1120, Wilmington, Mass.). The labeled18mer was made with the trityl group intact from a one micromole column.On completion of the synthesis the oligonucleotide was cleaved off thesupport with NH₄OH and purified by HPLC using an ABI RP-300 (C-8) column(4.6×220 mm) using a flow rate of 1.5 ml/min. and a solvent gradient of0.1M triethylammonium acetate-water pH 7.0 and acetonitrile, the tritylgroup was removed and the product was isolated by ethanol precipitation.

C3 spacer+Oligo seq. synthesis, no phosphorothioate group: An 18 baseoligonucleotide labeled with one or more C3 spacers (P/N 10-1913-90,Glen Research), at the 5′ position was made on an ABI model 394 DNAsynthesizer using standard phosphoramidite chemistry. The labeled 18merwas made with the trityl group intact from a one micromole column. Oncompletion of the synthesis the oligonucleotide was cleaved off thesupport with NH₄OH and purified by HPLC using an ABI RP-300 (C-8) column(4.6×220 mm) using a flow rate of 1.5 ml/min. and a solvent gradient of0.1M triethylammonium acetate-water pH 7.0 and acetonitrile, the tritylgroup was removed and the product was isolated by ethanol precipitation.

Protocol for oligo labeled with a 5′ phosphate: An 18 baseoligonucleotide labeled with a phosphate group at the 5′ position. Thiswas made on an ABI model 394 DNA synthesizer using standardphosphoramidite chemistry. The phosphate group was generated using aphosphoramidite obtained from Glen Research (P/N 10-1922-90) The labeled18mer was made from a one micromole column and on completion of thesynthesis the oligonucleotide was cleaved off the support with NH₄OH andpurified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using aflow rate of 1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile. The product was then isolated byethanol precipitation.

Protocol for oligo labeled with dual branching (doubler) linker labeledwith one or more C3 spacers: An 18 base oligonucleotide labeled with adual branching linkage followed by one or more C3 spacers at the 5′position was made on an ABI model 394 DNA synthesizer using standardphosphoramidite chemistry. The dual (doubler) branching (P/N 10-1920-90)and C3 spacer (P/N 10-1913-90) phosphoramidites were obtained from GlenResearch. The labeled 18mer was made with the trityl group intact usinga one micromole synthesis column. On completion of the synthesis theoligonucleotide was cleaved off the support with NH₄OH and purified byHPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using a flow rate of1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile, the trityl group was removed andthe product was isolated by ethanol precipitation.

Protocol for oligo labeled with trebler branching (trebler) linkerlabeled with one or more C3 spacers: An 18 base oligonucleotide labeledwith a trebler branching linkage followed by one or more C-3 spacers atthe 5′ position was made on an ABI model 394 DNA synthesizer usingstandard phosphoramidite chemistry. The trebler phosphoramidite (P/N10-1922-90) and C-3 spacer (P/N 10-1913-90) phosphoramidites wereobtained from Glen Research. The labeled 18mer was made with the tritylgroup intact using a one micromole synthesis column. On completion ofthe synthesis the oligonucleotide was cleaved off the support with NH₄OHand purified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm)using a flow rate of 1.5 ml/min. and a solvent gradient of 0.1Mtriethylammonium acetate-water pH 7.0 and acetonitrile, the trityl groupwas removed and the product was isolated by ethanol precipitation.

Protocol for oligo labeled with trebler branching linker end labeledwith phosphates (3 total phosphates): An 18 base oligonucleotide labeledwith a trebler branching linkage at the 5′ position followed byphosphorylation was made on an ABI model 394 DNA synthesizer usingstandard phosphoramidite chemistry. The trebler branching (P/N10-1922-90) and phosphorylating (P/N 10-1900-90) phosphoramidites wereobtained from Glen Research. The labeled 18mer was made using a onemicromole synthesis column. On completion of the synthesis theoligonucleotide was cleaved off the support with NH₄OH and purified byHPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using a flow rate of1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile. The product was isolated byethanol precipitation.

Protocol for oligo labeled with two generations of trebler branchinglinker end labeled with phosphates (9 total phosphates): An 18 baseoligonucleotide labeled with 2 additions of trebler branching linkagesat the 5′ position followed by phosphorylation was made on an ABI model394 DNA synthesizer using standard phosphoramidite chemistry. Thetrebler branching (P/N 10-1922-90) and phosphorylating (P/N 104900-90)phosphoramidites were obtained from Glen Research. The labeled 18mer wasmade using a one micromole synthesis column. On completion of thesynthesis the oligonucleotide was cleaved off the support with NH₄OH andpurified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using aflow rate of 1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile. The product was isolated byethanol precipitation.

Protocol for oligo labeled with one or more C-3 spacer containing a 3′phosphorothioate linkage: An 18 base oligonucleotide labeled with one ormore C-3 spacers at the 5′ position was made on an ABI model 394 DNAsynthesizer using standard phosphoramidite chemistry. The 3′phosphorothioate linkage was made using standard methods withsulfurizing reagent (TETD P/N 401267 (Applied Biosystems, Foster City,Calif.). The C3 spacer phosphoramidite was obtained from Glen Research(P/N 10-1913-90). The labeled 18mer was made with the trityl groupintact from a one micromole synthesis column. On completion of thesynthesis the ol_(ig)onucleotide was cleaved off the support with NH₄OHand purified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm)using a flow rate of 1.5 ml/min. and a solvent gradient of 0.1Mtriethylammonium acetate-water pH 7.0 and acetonitrile, the trityl groupwas removed and the product was isolated by ethanol precipitation. Note:To synthesize more than one phosphorothioate linkage or to place thislinkage anywhere in the 18-mer oligonucleotide chain, oxidize using thesulfurizing reagent at these position(s).

Example 2 PCR and Sequencing Reactions

PCR and Sequencing Reactions Using a Chemically-Enhanced Primer with aTerminal 3′ Phosphorothioate (PS) Linkage

PCR Amplification

PCR reactions were carried out in the following 10 μL solution: tosequence amplicon RSA000013703, 1 μL 4 ng/μL gDNA, M13-tagged primers(0.8 uM each): 1.5 μL of TGTAAAACGACGGCCAGTTTGATGGGCTCAGCAACAGGT (SEQ IDNO:1, gnl|Probe|1292199b) and CAGGAAACAGCTATGACCCCACTGCTTGCGTTTCTTCCTG(SEQ ID NO:2, gnl|Probe|11292199c), 5 μL of BigDye Direct PCR Master Mix(P/N 4458699, Applied Biosystems) and 2.5 μL water. PCR was carried outon a Veriti™ 96-well thermal cycler (P/N 4375786, Applied Biosystems).with the following thermal cycling conditions: 95° C. for 10 minutes,then 35 cycles of 96° C. for 3 seconds, 62° C. for 15 seconds, and 68°C. for 30 sec. followed by 72° C. for 2 min. and 4° C. hold. To confirmamplification a 10 μL sample was analyzed on an agarose gel. A bandconsistent with the expected 626 bp amplicon was observed.

Amplicon RSA000013703 SEQ ID NO:3, template ZC is shown below withprimer binding sites for PCR forward primer, and PCR reverse primer(reverse complement) underlined.

CAGGAAACAGCTATGACCCCACTGCTTGCGTTTCTTCCTGTTTTAATCCCACTTTCAATGAAGTGTGTATTTGAAATAAATGGCTCATGAGTTAATCACATCTTTATATATCCTAAGATGTATTACAAAGGCTTCCATAACACTTGTCTATAGTAAGCCACTCATTTCTATAATTTTTCTTTCAATAAACTCAATCTTTGTAATACAGAAATTAACCTTCTGGGTTGTTTTTGTTCAAGATCTTCAGTTTGATTTGCCCCTTGGTTGATCTGTTTTTCCCATCGCTGAACTGGTTCCCATAATCACACACCTTTGCTTTTCATTTCCACAGATCAAGGAATCAACATTTACCGAAAGCCACCCATCTACAAACAGCATGGTAAAACCCGCTTTCCTCCGCGTAGCTTTTAAATAGCAAAGTCAGCTGAACTTCTCCTTGCTGTCCTCTGAAAGGCTTTTCCTGCTGCTGCTTTTGAGAGTAAAACTGGGGCATCCAGCATATTATGCCTTTCTGGTCTACTAAGATGTAAATATTGTAAAATTGATTCTCCTGGATGGAGAGACTTAGCTTGATTAGAAAGCTTCTAACCTGTTGCTGAGCCCATCAAACTGGCCGTCGTTTTACA.

Sequencing Workflow with a Chemically Enhanced Primer Having aPhosphorothioate (PS) Linkage

Sequencing reaction prepared with the BigDye Direct Cycle Sequencing Kit(24 reactions, P/N 4458689, Applied Biosystems): 10 μL of the PCRamplification reaction was mixed with 2 μL BigDye® Direct SequencingMaster Mix (P/N 4458701, Applied Biosystems), and 1 μL BigDye Direct M13Forward Primer (P/N 4458692 Applied Biosystems) or BigDye Direct M13Reverse Primer 4458695, Applied Biosystems). The BigDye Direct Primershave the terminal 5′ NCM and a terminal 3′ PS. The cycle sequencingreaction was carried out on a Verti 96-well thermal cycler at 37° C. 15min. at which point excess PCR amplification primers were digested bythe ExoI nuclease (contained in the BigDye Direct Sequencing MasterMix), then 80° C. 2 min, 96° C. 1 min. followed by 25 cycles, of 96° C.10 seconds, 50° C. 5 seconds, and 60° C. 1 minute 15 seconds and 4° C.hold. The Sequencing primer contained a terminal 5′ Negatively ChargedMoiety (NCM) and a terminal 3′ phosphorothioate group indicated by anasterisk: M13 forward primer (1 μM) (NCM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4) or M13 reverse primer (1 μM) (NCM-CAGGAAACAGCTATGAC*C) (SEQ IDNO:5). FIG. 3H provides the structure of the NCM. FIG. 11 is aelectropherogram (see U.S. Ser. No. 61/407,899, filed Oct. 28, 2011 andU.S. Ser. No. 61/408,553, filed Oct. 29, 2011), of a chemically-enhancedprimer (FIG. 3H) with a terminal 3′ PS linkage and ZC as the template.The sequence of ZC shows high resolution at base 1 from the primer.

Example 3

PCR reactions were carried out in the following 10 μL solution: tosequence amplicon RSA000317141 (Template Seq01, 545 bp), 1 μL 4 ng/μLgDNA, M13-tagged primers (0.8 uM each): 1.5 μL ofTGTAAAACGACGGCCAGTGCTGCCTCTGATGGCGGAC (SEQ ID NO:6, forward,gnl|Probe|11204459b) and CAGGAAACAGCTATGACCGCCACACTCTGGAGCTGGACA (SEQ IDNO:7, reverse, gnl|Probe|11204459c), 5 μL of BigDye Direct PCR MasterMix (P/N 4458699, Applied Biosystems) and 2.5 μL water. PCR was carriedout on a Veriti™ 96-well thermal cycler (P/N 4375786, AppliedBiosystems). with the following thermal cycling conditions: 95° C. for10 minutes, then 35 cycles of 96° C. for 3 seconds, 62° C. for 15seconds, and 68° C. for 30 sec. followed by 72° C. for 2 min. and 4° C.hold. To confirm amplification a 10 μL sample was analyzed on an agarosegel. A band consistent with the expected 545 bp amplicon was observed.

Amplicon RSA000317141 SEQ ID NO:8, template Seq01 (FIG. 3I) is shownbelow with primer binding sites for PCR forward primer, and PCR reverseprimer (reverse complement) underlined.

TGTAAAACGACGGCCAGTGCTGCCTCTGATGGCGGACGGGGGTGTGGTCCTGGGACTCGTGGTCAGGGCTGGTCTGTGTGGAATGCTGATCCTTCTCTTCCCCAATCTACCTGTGTCAGTTCCCTCCTTTTCTATTTTCTCTTCCCTGCAGATGTCAAGCCCTCCAACATCCTAGTCAACTCCCGTGGGGAGATCAAGCTCTGTGACTTTGGGGTCAGCGGGCAGCTCATCGACTCCATGGCCAACTCCTTCGTGGGCACAAGGTCCTACATGTCGGTATGAACAGAAGTTTCCATTGCTTGAGCTTCTTGTACGGTCAGGGAGAGGAGCCCAGTGGGTGCCTTTCCTGTGGAGCCAGAGTCTTGTGCTGGGTAGGGGACAAGAAGTGAGGGAGGAGGCACAGTGCTCTGCCCTGAGGAGATGAAGTTGAATGGGAAGATGGTCTTGGTCTTTCTTAGGCCTTGGAGCATAACTGGGATATTGGGGCCTTGACTCACTGAAAGGACTGTCCAGCTCCAGAGTGTGGCGGTCATAGCTGTTTCCTG.

Sequencing workflow with a chemically enhanced primer having aphosphorothioate (PS) linkage.

Sequencing reaction prepared with the BigDye Direct Cycle Sequencing Kit(24 reactions, P/N 4458689, Applied Biosystems): 10 μL of the PCRamplification reaction was mixed with 2 μL BigDye® Direct SequencingMaster Mix (P/N 4458701, Applied Biosystems), and 1 μL BigDye Direct M13Forward Primer (P/N 4458692 Applied Biosystems) or BigDye Direct M13Reverse Primer 4458695, Applied Biosystems). The BigDye Direct Primershave the terminal 5′ NCM and a terminal 3′ PS. The cycle sequencingreaction was carried out on a Verti 96-well thermal cycler at 37° C. 15min. at which point excess PCR amplification primers were digested bythe ExoI nuclease (contained in the BigDye Direct Sequencing MasterMix), then 80° C. 2 min, 96° C. 1 min. followed by 25 cycles, of 96° C.10 seconds, 50° C. 5 seconds, and 60° C. 1 minute 15 seconds and 4° C.hold. The Sequencing primer contained a terminal 5′ Negatively ChargedMoiety (NCM) and a terminal 3′ phosphorothioate group indicated by anasterisk: M13 forward primer (1 μM) (NCM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4) or M13 reverse primer (1 μM) (NCM-CAGGAAACAGCTATGAC*C) (SEQ IDNO:5). FIG. 16 (see U.S. Ser. No. 61/408,553, filed Oct. 29, 2011),provides a electropherogram of a chemically-enhanced primer with aterminal 3′ PS linkage and RSA000317141 as the template (FIG. 3J). Thesequence of RSA000317141 shows high resolution at base 1 from theprimer.

Example 4

PCR and Sequencing Reactions with or without PS linkage Standard PCRAmplification: PCR reactions were carried out in the following 10 μLsolution: for example to sequence amplicon RSA0003176671 μL 10 ng/μLgDNA, primers (0.8 uM each), 1.5 μL ofTGTAAAACGACGGCCAGTGGCTCCTGGCACAAAGCTGG (gnl|Probe|11172813b, forward,SEQ ID NO:9) and CAGGAAACAGCTATGACCTGCATCTCATTCTCCAGGCTTC(gnl|Probe|11172813c, reverse, SEQ ID NO:10),), 5 μL of AmpliTaq Gold®PCR Master Mix (P/N 4318739, Applied Biosystems), 1.6 μL 50% glyceroland 0.9 μL water. PCR was carried using a Gold-plated 96-Well GeneAmp®PCR System 9700 (P/N 4314878, Applied Biosystems). Thermal cyclingconditions: 96° C. 5 minutes, then 40 cycles of 94° C. 30 seconds, 60°C. 45 seconds, and 72° C. 45 sec. followed by 72° C. 2 min. and 4° C.hold. A 5 μL aliquot was analyzed on an agarose gel. A band consistentwith the expected 630 bp amplicon was observed.

PCR clean-up: The 10 μL of PCR amplification reaction was mixed with 2μL of ExoSAP-IT® nuclease (P/N 78250, Affymetrix, Santa Clara, Calif.)and incubated on a Gold-plated 96-Well GeneAmp® PCR System 9700 (P/N4314878, Applied Biosystems) at 37° C. 30 minutes followed by 80° C. for15 minutes (inactivated the nuclease.

Sequencing Workflow with a Chemically Enhanced Primer withoutPhosphorothioate Linkage

A sequencing reaction was prepared with the BigDye® Terminator v3.1Cycle Sequencing Kit (24 reactions, P/N 4337454, Applied Biosystems): 2μL of the PCR amplification reaction treated with ExoSAP-IT was mixedwith 4 μL BigDye® Terminator v3.1 Cycle Sequencing Kit Master Mix (P/N4337454, Applied Biosystems), 1 μL Sequencing primerschemically-enhanced with terminal 5′ NCM and with or without a terminal3′ phosphorothioate linkage, (NCM-M13 forward and NCM-M13 reverseprimer) and 3 μL water. The cycle sequencing reaction was carried out at96° C. 1 min. followed by 25 cycles, 96° C. 10 sec. 50° C. 5 sec, 60° C.1 min. 15 sec followed by 4° C. hold. For example, the sequencing primeris M13 forward primer (1 μM) containing a terminal 3′ phosphorothioate(PS) group indicated by an asterisk (NGM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4), M13 reverse primer (1 μM) (NGM-CAGGAAACAGCTATGAC*C) (SEQ ID NO:5)or without PS, M13 forward primer (1 μM) (NGM-TGTAAAACGACGGCCAGT) (SEQID NO:11), M13 reverse primer (1 μM) (NGM-CAGGAAACAGCTATGACC) (SEQ IDNO:12). FIGS. 4 and 8 provide electropherograms (see U.S. Ser. No.61/407,899, filed Oct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct.29, 2011), of chemically-enhanced sequencing primers (FIGS. 3A and 3F,respectively), with terminal 5′ negatively charged moieties, without PSand RSA000317667 as the template. The sequences for RSA000317667 showhigh resolution at base 1 from the primer.

Example 5 Capillary Electrophoresis Sample Preparation and Detection

The amplified samples are analyzed by methods that resolve nucleobasesequences as would be known to one of skill in the art. For example,capillary electrophoresis can be used following the instrumentmanufactures directions. BigDye XTerminator Purification Kit (AppliedBiosystems, P/N 4376486) can be used in cycle sequencing clean up toprevent the co-injection of un-incorporated dye-labeled terminators,dNTPs and salts with dye-labeled extension products into a capillaryelectrophoresis DNA analyzer. Briefly, 13 μL sequencing reaction mixturewas combined with 45 μL SAM Solution and 10 μL XTerminator Solution.After vortexing the sample plate at 1800 rpm for 20 minutes, spin theplate at 1000×g for 2 minutes. To each well was added 30 μL of 70%ethanol and the plate was centrifuged at 1650×g for 15 minutes. Thesolution was removed by inverting the plate onto a paper towel andcentrifuging at 180×g for 1 minute. The precipitated sequencing reactionwas then dissolved in 10 μL of 50 μM EDTA and loaded onto an AB 3500xLGenetic Analyzer equipped with a 50 cm capillary array (AppliedBiosystems, Foster City, Calif.).

Example 6 Capillary Electrophoresis Methods and Analysis

Capillary electrophoresis (CE) was performed on the current AppliedBiosystems instruments, for example the Applied Biosystems 3500xlGenetic Analyzer, using the dye set Z as described the instrument's UserGuide. There are ShortReadSeq_BDX_POP7, RapidSeq_BDX_POP7,FastSeq_BDX_POP7, StdSeq_BDX_POP7 run modules. For example,BDxFastSeq50_POP7xl_(—)1 parameters were: oven temperature: 60 C, sampleinjection for 5 sec at 1.6 kV and electrophoresis at 13.4 kV for 2520sec in Performance Optimized Polymer (POP-7™ polymer) with a runtemperature of 60° C. Variations in instrument parameters, e.g.injection conditions, were different on other CE instruments such as the3500 or 3730xl Genetic Analyzers. The data were collected using versionsthe Applied Biosystems Data Collection Software specific to thedifferent instruments, such as 3500 Data Collection Software v1.0. Thesequence traces were analyzed by Applied Biosystems KB™ BasecallerSoftware v1.4.1 with KB_(—)3500_POP7—BDTv3direct.bcc andKB_(—)3500_POP7—BDTv3direct.mob to determine the correct base calls.

Example 7 Sequencing of Plasmid DNA or PCR Amplified Exo/SAP Treated DNA

200 ng of pGem plasmid DNA or 10 ng of Exo/SAP treated PCR product mixedwith BigDye Terminator v3.1 Ready Reaction reagent and sequencing primerhaving an NCM composition without PS was reacted in a cycle sequencingreaction: 95 C/1 min and 25 cycles of (96 C/1 min, 50 C/5 sec, 60 C/1min 15 sec). The reaction was cleaned up using BigDye XTerminator Kit(Applied Biosystems) and electrophrosesed on the 3500 with POP7 polymer.The electropherogram of the sequenced pGEM plasmid sequenced using thesequencing primer having NCM on the 5′ indicated clear sequence withinfive base pairs of the primer (data not shown). The electropherogram ofthe sequenced pGEM plasmid sequenced using the sequencing primer havingNCM on the 5′ and nuclease resistance linkage on the 3′ end indicatedclear sequence five base pairs after the primer (data not shown). Theelectropherogram of the Exo/SAP treated PCR product sequenced using thesequencing primer with NCM on the 5′ and nuclease resistance linkage onthe 3′ end indicated clean sequence reads six bases after the primer(data not shown).

Those skilled in the art understand that the detection techniquesemployed are generally not limiting. Rather, a wide variety of detectionmeans are within the scope of the disclosed methods and kits, providedthat they allow the presence or absence of an amplicon to be determined.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are made only by way of example and are not intended tolimit the scope of the invention. What has been disclosed herein hasbeen provided for the purposes of illustration and description. It isnot intended to be exhaustive or to limit what is disclosed to theprecise forms described. Many modifications and variations will beapparent to the practitioner skilled in the art. What is disclosed waschosen and described in order to best explain the principles andpractical application of the disclosed embodiments of the art described,thereby enabling others skilled in the art to understand the variousembodiments and various modifications that are suited to the particularuse contemplated. It is intended that the scope of what is disclosed bedefined by the following claims and their equivalence.

1. A chemically-enhanced primer comprising: an oligonucleotide sequencehaving at least one nuclease-resistant linkage or no nuclease-resistantlinkages and a negatively charged moiety (NCM) wherein the NCM isselected from a (Cn) spacer or a branched (Cn) spacer, wherein n can beany integer from 1 to
 9. 2. The chemically-enhanced primer of claim 1,wherein when the oligonucleotide comprises at least onenuclease-resistant linkage, the at least one nuclease-resistant linkageis at a terminal 3′ end of the oligonucleotide sequence.
 3. Thechemically-enhanced primer of claim 1, wherein the NCM comprises aplurality of negatively charged moieties, and wherein the NCM isattached to a terminal 5′ end or within the oligonucleotide sequence.4.-5. (canceled)
 6. The chemically-enhanced primer of claim 3, whereinwhen n equals 3 the NCM comprises a (C3)_(x) spacer, wherein x equals atleast 5, at least 6, at least 8, at least 9, at least 10, at least 15 atleast 18 or more C3 spacers in a linear arrangement or [(C3)_(x)]z,wherein z equals 2 or 3 in a branched arrangement.
 7. Thechemically-enhanced primer of claim 6, wherein the branched (C3)_(x) canbe a doubler or a trebler.
 8. The chemically-enhanced primer of claim 1,wherein the oligonucleotide sequence comprises a universal primersequence or a gene-specific primer sequence.
 9. The chemically-enhancedprimer of claim 8, wherein the universal primer sequence is selectedfrom M13, US1, T7, SP6, and T3.
 10. The chemically-enhanced primer ofclaim 1, wherein the chemically enhanced primer is resistant todigestion by a nuclease.
 11. The chemically-enhanced primer of claim 1,wherein the NCM can be a combination of [C3]_(x) and [C6]_(y) spacers,wherein y equals an integer from 1 to
 20. 12. The chemically-enhancedprimer of claim 1, further comprising a dye-label.
 13. Thechemically-enhanced primer of claim 12, wherein the dye-label isattached to the NCM or the oligonucleotide sequence.
 14. Thechemically-enhanced primer of claim 13, wherein the dye-label is afluorescent dye-label.
 15. The chemically-enhanced primer of claim 10,wherein the nuclease is selected from exonuclease I, Exo III, Pfu andDNA pol I. 16-35. (canceled)
 36. A method of preparing DNA forsequencing, comprising the steps of: amplifying the DNA under conditionsto produce amplification reaction products, the amplification reactionproducts comprising excess amplification primer; contacting saidamplification reaction products with a reaction mixture comprising anuclease and a chemically-enhanced primer, whereby the excessamplification primer is degraded by the nuclease and thechemically-enhanced primer is essentially non-degraded, wherein thechemically-enhanced primer comprises at least one negative chargedmolecule (NCM) wherein the NCM is selected from a (Cn) spacer or abranched (Cn) spacer, wherein n can be any integer from 1 to 9, and anoligonucleotide sequence having at least one nuclease-resistant linkageor no nuclease-resistant linkages. 37-39. (canceled)
 40. A method forsequencing DNA, comprising the steps of: a. amplifying DNA in a firstreaction mixture comprising nuclease-sensitive amplification primers toform amplified DNA; b. contacting the first reaction mixture of theamplifying step with a second reaction mixture comprising a nuclease anda chemically-enhanced primer, whereby the nuclease sensitiveamplification primers are degraded by the nuclease; c. inactivating thenuclease; and d. reacting the amplified DNA in a sequencing reactionwherein the chemically-enhanced primer primes the sequencing reaction.41. The method of claim 40, further comprising: a. obtaining sequencingresults based on the sequencing reaction; and b. determining anucleotide base sequence of the amplified DNA based on the results.42.-43. (canceled)
 44. The method of claim 40, wherein thechemically-enhanced primer comprises an oligonucleotide sequence havingat least one nuclease-resistant linkage or no nuclease-resistantlinkages, a negative charged molecule (NCM) selected from a (Cn) spaceror a branched (Cn) spacer, wherein n can be any integer from 1 to 9.45-51. (canceled)
 52. The method of claim 40, wherein the secondreaction mixture further comprises a polymerase, deoxynucleotidetriphosphates, dideoxynucleotide triphosphates and a dye-label. 53.-58.(canceled)
 53. A kit comprising: a chemically-enhanced primer,comprising at least one negative charged molecule (NCM) wherein the NCMis selected from a (Cn) spacer or a branched (Cn) spacer, wherein n canbe any integer from 1 to 9, and an oligonucleotide sequence having atleast one nuclease-resistant linkage or no nuclease-resistant linkages.60.-61. (canceled)
 62. The chemically-enhanced primer of claim 1, havingthe structure of formula:


63. The chemically-enhanced primer of claim 1, having the structure ofone of the following formulae: